Optical Microscopy Using the Faraday Effect Reveals in Situ Magnetization Dynamics of Magnetic Nanoparticles in Biological Samples.

The study of exogenous and endogenous nanoscale magnetic material in biology is important for developing biomedical nanotechnology as well as for understanding fundamental biological processes such as iron metabolism and biomineralization. Here, we exploit the magneto-optical Faraday effect to probe intracellular magnetic properties and perform magnetic imaging, revealing the location-specific magnetization dynamics of exogenous magnetic nanoparticles within cells. The opportunities enabled by this method are shown in the context of magnetic hyperthermia; an effect where local heating is generated in magnetic nanoparticles exposed to high-frequency AC magnetic fields. Magnetic hyperthermia has the potential to be used as a cellular-level thermotherapy for cancer, as well as for other biomedical applications that target heat-sensitive cellular function. However, previous experiments have suggested that the cellular environment modifies the magnetization dynamics of nanoparticles, thus dramatically altering their heating efficiency. By combining magneto-optical and fluorescence measurements, we demonstrate a form of biological microscopy that we used here to study the magnetization dynamics of nanoparticles in situ, in both histological samples and living cancer cells. Correlative magnetic and fluorescence imaging identified aggregated magnetic nanoparticles colocalized with cellular lysosomes. Nanoparticles aggregated within these lysosomes displayed reduced AC magnetic coercivity compared to the same particles measured in an aqueous suspension or aggregated in other areas of the cells. Such measurements reveal the power of this approach, enabling investigations of how cellular location, nanoparticle aggregation, and interparticle magnetic interactions affect the magnetization dynamics and consequently the heating response of nanoparticles in the biological milieu.

Fabrication and Characterisation of Hydrogels with Reversible Wrinkled Surfaces for Limbal Study and Reconstruction.

In the biomedical field, there is a demand for the development of novel approaches for the investigation of optical epithelial anatomical features with biomimetic materials. These materials are not only required to replicate structures but also enable dynamic modelling for disease states such as limbal stem cell deficiency and ageing. In the present study, the effective generation of reversible wrinkled polydimethylsiloxane (PDMS) substrates was undertaken to mimic the undulating anatomy of the limbal epithelial stem cell niche. This undulating surface pattern was formed through a dual treatment with acid oxidation and plasma using an innovatively designed stretching frame. This system enabled the PDMS substrate to undergo deformation and relaxation, creating a reversible and tuneable wrinkle pattern with cell culture applications. The crypt-like pattern exhibited a width of 70-130 µm and a depth of 17-40 µm, resembling the topography of a limbal epithelial stem cell niche, which is characterised by an undulating anatomy. The cytocompatibility of the patterned substrate was markedly improved using a gelatin methacrylate polymer (GelMa) coating. It was also observed that these wrinkled PDMS surfaces were able to dictate cell growth patterns, showing alignment in motile cells and colony segregation in colony-forming cells when using human and porcine limbal cells, respectively.

Magnetic Ion Channel Activation (MICA)-Enabled Screening Assay: A Dynamic Platform for Remote Activation of Mechanosensitive Ion Channels.

This study reports results of a mechanical platform-based screening assay (MICA) to evaluate the remote activation of mechanosensitive ion channels. Here, we studied ERK pathway activation and the elevation in intracellular Ca2+ levels in response to the MICA application using the Luciferase assay and Fluo-8AM assay, respectively. Functionalised magnetic nanoparticles (MNPs) targeting membrane-bound integrins and mechanosensitive TREK1 ion channels were studied with HEK293 cell lines under MICA application. The study demonstrated that active targeting of mechanosensitive integrins via RGD (Arginylglycylaspartic acid) motifs or TREK1 (KCNK2, potassium channel subfamily K member 2) ion channels can stimulate the ERK pathway and intracellular calcium levels compared to non-MICA controls. This screening assay offers a powerful tool, which aligns with existing high-throughput drug screening platforms for use in the assessment of drugs that interact with ion channels and influence ion channel-modulated diseases.

Remote Activation of Mechanotransduction via Integrin Alpha-5 via Aptamer-Conjugated Magnetic Nanoparticles Promotes Osteogenesis.

Bone regeneration and repair are complex processes in the adult skeleton, and current research has focused on understanding and controlling these processes. Magnetic nanoparticle (MNP)-based platforms have shown potential in tissue engineering and regenerative medicine through the use of magnetic nanomaterials combined with remotely applied dynamic fields. Previous studies have demonstrated the ability of MNP-induced mechanoactivation to trigger downstream signaling and promote new bone formation. In this study, we aimed to compare the osteogenic induction achieved using the mechanoreceptor targets, Piezo1, Fzd1, Fzd2, and integrin alpha-5. We compared the binding efficacy of different types of agonists (antibodies vs. aptamers) to these receptors. Moreover, we optimized the aptamer concentration (2.5, 5, and 10 µg/mg) for the selected receptor to determine the optimum concentration for promoting bone formation. Our data demonstrated that the mechanoactivation of integrins (CD49e) significantly upregulated the RUNX2 and LEF1 genes compared to other selected receptors. Furthermore, comparing the mechanoactivation of cells using MNPs conjugated with CD49e antibodies and aptamers revealed that MNP-aptamers significantly enhanced the upregulation of LEF1 genes. This suggests that aptamer-mediated mechanoactivation is a promising alternative to antibody-mediated activation. Finally, our results showed that the concentration of the aptamer loaded onto the MNPs strongly influenced the mechanoactivation of the cells. These findings provide valuable insights into the use of MNP platforms for bone regeneration and highlight the potential of aptamers in promoting signaling pathways related to bone formation. The novelty of our study lies in elucidating the unique advantages of aptamers in mediating mechanoactivation, presenting a promising avenue for advancing bone regenerative strategies.

Magnetic activation of TREK1 triggers stress signalling and regulates neuronal branching in SH-SY5Y cells.

TWIK-related K+ 1 (TREK1) is a potassium channel expressed in the nervous system with multiple functions including neurotransmission and is a prime pharmacological target for neurological disorders. TREK1 gating is controlled by a wide range of external stimuli including mechanical forces. Previous work has demonstrated that TREK1 can be mechano-activated using magnetic nanoparticles (MNP) functionalised with antibodies targeted to TREK1 channels. Once the MNP are bound, external dynamic magnetic fields are used to generate forces on the TREK channel. This approach has been shown to drive cell differentiation in cells from multiple tissues. In this work we investigated the effect of MNP-mediated TREK1 mechano-activation on early stress response pathways along with the differentiation and connectivity of neuronal cells using the model neuronal cell line SH-SY5Y. Results showed that TREK1 is well expressed in SH-SY5Y and that TREK1-MNP initiate c-Myc/NF-κB stress response pathways as well as Nitrite production after magnetic stimulation, indicative of the cellular response to mechanical cues. Results also showed that TREK1 mechano-activation had no overall effect on neuronal morphology or expression of the neuronal marker ßIII-Tubulin in Retinoic Acid (RA)/Brain-derived Neurotrophic factor (BDNF) differentiated SH-SY5Y but did increase neurite number. These results suggest that TREK1 is involved in cellular stress response signalling in neuronal cells, which leads to increased neurite production, but is not involved in regulating RA/BDNF mediated neuronal differentiation.

Immobilization of Wnt Fragment Peptides on Magnetic Nanoparticles or Synthetic Surfaces Regulate Wnt Signaling Kinetics.

Wnt signaling plays an important role in embryogenesis and adult stem cell homeostasis. Its diminished activation is implicated in osteoporosis and degenerative neural diseases. However, systematic administration of Wnt-signaling agonists carries risk, as aberrantly activated Wnt/ß-catenin signaling is linked to cancer. Therefore, technologies for local modulation and control of Wnt signaling targeted to specific sites of disease or degeneration have potential therapeutic value in the treatment of degenerative diseases. We reported a facile approach to locally activate the canonical Wnt signaling cascade using nanomagnetic actuation or ligand immobilized platforms. Using a human embryonic kidney (HEK293) Luc-TCF/LEF reporter cell line, we demonstrated that targeting the cell membrane Wnt receptor, Frizzled 2, with peptide-tagged magnetic nanoparticles (MNPs) triggered canonical Wnt signaling transduction when exposed to a high-gradient, time-varying magnetic field, and the induced TCF/LEF signal transduction was shown to be avidity-dependent. We also demonstrated that the peptide retained signaling activity after functionalization onto glass surfaces, providing a versatile platform for drug discovery or recreation of the cell niche. In conclusion, these results showed that peptide-mediated Wnt signaling kinetics depended not only on ligand concentration but also on the presentation method of the ligand, which may be further modulated by magnetic actuation. This has important implications when designing future therapeutic platforms involving Wnt mimetics.

Magnetic Mechanoactivation of Wnt Signaling Augments Dopaminergic Differentiation of Neuronal Cells.

Wnt signaling is a key developmental pathway that regulates dopaminergic progenitor cell proliferation and differentiation during neuronal development. This makes Wnt signaling an important therapeutic target for neurodegenerative conditions such as Parkinson's disease. Wnt signaling can be modulated using peptides such as UM206, which bind to the Wnt receptor Frizzled. Previous work has demonstrated remote activation of the Wnt pathway through Frizzled using peptide-functionalized magnetic nanoparticles (MNPs) with magnetic field stimulation. Using this technology, Wnt signaling is remotely activated in the neuronal cell line SH-SY5Y, and the phenotypic response to stimulation is assessed. Results indicate ß-catenin translocalization and activation of TCF/LEF responsive transcription in response to MNP and magnetic fields, which result in dopaminergic marker expression when synergistically combined with differentiation factors retinoic acid and the phorbol ester phorbol 12-myristate 13-acetate. This approach is translated into ex vivo postnatal rat brain slices modeling the developing nigrostriatal pathway. Dopaminergic marker expression is maintained in MNP-labeled SH-SY5Y cells after injection and magnetic stimulation. These results demonstrate the translational value of remote control of signal transduction for controlling neuronal precursor cell behavior and highlight the potential applications for controlled cell differentiation as part of cell therapies for neurodegenerative disease.

Magnetic ion channel activation of TREK1 in human mesenchymal stem cells using nanoparticles promotes osteogenesis in surrounding cells.

Magnetic ion channel activation technology uses superparamagnetic nanoparticles conjugated with targeting antibodies to apply mechanical force directly to stretch-activated ion channels on the cell surface, stimulating mechanotransduction and downstream processes. This technique has been reported to promote differentiation towards musculoskeletal cell types and enhance mineralisation. Previous studies have shown how mesenchymal stem cells injected into a pre-mineralised environment such as a foetal chick epiphysis, results in large-scale osteogenesis at the target site. However, the relative contributions of stem cells and surrounding host tissue has not been resolved, that is, are the mesenchymal stem cells solely responsible for the observed mineralisation or do mechanically stimulated mesenchymal stem cells also promote a host-tissue mineralisation response? To address this, we established a novel two-dimensional co-culture assay, which indicated that magnetic ion channel activation stimulation of human mesenchymal stem cells does not significantly promote migration but does enhance collagen deposition and mineralisation in the surrounding cells. We conclude that one of the important functions of injected human mesenchymal stem cells is to release biological factors (e.g., cytokines and microvesicles) which guide the surrounding tissue response, and that remote control of this signalling process using magnetic ion channel activation technology may be a useful way to both drive and regulate tissue regeneration and healing.

Remote regulation of magnetic particle targeted Wnt signaling for bone tissue engineering.

Wnt signaling is critically involved in the differentiation of human Mesenchymal Stem Cells (hMSC). Wnt proteins therefore have considerable therapeutic value, but are expensive and difficult to produce. UM206 is a synthetic peptide and ligand for the Wnt receptor Frizzled. Attachment of UM206 to magnetic nanoparticles (MNP) enables the ligand-MNP complex to be manipulated using magnetic fields, allowing control of Frizzled stimulation. Using this approach, Wnt signaling was activated in hMSC which resulted in Frizzled clustering, ß-catenin translocalization and activation of TCF/LEF responsive transcription. During osteogenesis, UM206-MNP initiated localized mineralized matrix formation. Injection and magnetic stimulation of UM206-MNP-labeled MSC in ex vivo chick femurs resulted in increased mineralization which acted synergistically with addition of bone morphogenic protein 2 (BMP2) releasing micro-particles. As this facilitates external control over signal transduction, conjugated MNP technology has applications both as a research tool and for regulating tissue formation in clinical cell therapies.

Immobilized WNT Proteins Act as a Stem Cell Niche for Tissue Engineering.

The timing, location, and level of WNT signaling are highly regulated during embryonic development and for the maintenance of adult tissues. Consequently the ability to provide a defined and directed source of WNT proteins is crucial to fully understand its role in tissue development and to mimic its activity in vitro. Here we describe a one-step immobilization technique to covalently bind WNT3A proteins as a basal surface with easy storage and long-lasting activity. We show that this platform is able to maintain adult and embryonic stem cells while also being adaptable for 3D systems. Therefore, this platform could be used for recapitulating specific stem cell niches with the goal of improving tissue engineering.

Highly efficient delivery of functional cargoes by the synergistic effect of GAG binding motifs and cell-penetrating peptides.

Protein transduction domains (PTDs) are powerful nongenetic tools that allow intracellular delivery of conjugated cargoes to modify cell behavior. Their use in biomedicine has been hampered by inefficient delivery to nuclear and cytoplasmic targets. Here we overcame this deficiency by developing a series of novel fusion proteins that couple a membrane-docking peptide to heparan sulfate glycosaminoglycans (GAGs) with a PTD. We showed that this GET (GAG-binding enhanced transduction) system could deliver enzymes (Cre, neomycin phosphotransferase), transcription factors (NANOG, MYOD), antibodies, native proteins (cytochrome C), magnetic nanoparticles (MNPs), and nucleic acids [plasmid (p)DNA, modified (mod)RNA, and small inhibitory RNA] at efficiencies of up to two orders of magnitude higher than previously reported in cell types considered hard to transduce, such as mouse embryonic stem cells (mESCs), human ESCs (hESCs), and induced pluripotent stem cells (hiPSCs). This technology represents an efficient strategy for controlling cell labeling and directing cell fate or behavior that has broad applicability for basic research, disease modeling, and clinical application.

Remote activation of the Wnt/ß-catenin signalling pathway using functionalised magnetic particles.

Wnt signalling pathways play crucial roles in developmental biology, stem cell fate and tissue patterning and have become an attractive therapeutic target in the fields of tissue engineering and regenerative medicine. Wnt signalling has also been shown to play a role in human Mesenchymal Stem Cell (hMSC) fate, which have shown potential as a cell therapy in bone and cartilage tissue engineering. Previous work has shown that biocompatible magnetic nanoparticles (MNP) can be used to stimulate specific mechanosensitive membrane receptors and ion channels in vitro and in vivo. Using this strategy, we determined the effects of mechano-stimulation of the Wnt Frizzled receptor on Wnt pathway activation in hMSC. Frizzled receptors were tagged using anti-Frizzled functionalised MNP (Fz-MNP). A commercially available oscillating magnetic bioreactor (MICA Biosystems) was used to mechanically stimulate Frizzled receptors remotely. Our results demonstrate that Fz-MNP can activate Wnt/ß-catenin signalling at key checkpoints in the signalling pathway. Immunocytochemistry indicated nuclear localisation of the Wnt intracellular messenger ß-catenin after treatment with Fz-MNP. A Wnt signalling TCF/LEF responsive luciferase reporter transfected into hMSC was used to assess terminal signal activation at the nucleus. We observed an increase in reporter activity after treatment with Fz-MNP and this effect was enhanced after mechano-stimulation using the magnetic array. Western blot analysis was used to probe the mechanism of signalling activation and indicated that Fz-MNP signal through an LRP independent mechanism. Finally, the gene expression profiles of stress response genes were found to be similar when cells were treated with recombinant Wnt-3A or Fz-MNP. This study provides proof of principle that Wnt signalling and Frizzled receptors are mechanosensitive and can be remotely activated in vitro. Using magnetic nanoparticle technology it may be possible to modulate Wnt signalling pathways and thus control stem cell fate for therapeutic purposes.

Remotely Activated Mechanotransduction via Magnetic Nanoparticles Promotes Mineralization Synergistically With Bone Morphogenetic Protein 2: Applications for Injectable Cell Therapy.

Bone requires dynamic mechanical stimulation to form and maintain functional tissue, yet mechanical stimuli are often lacking in many therapeutic approaches for bone regeneration. Magnetic nanoparticles provide a method for delivering these stimuli by directly targeting cell-surface mechanosensors and transducing forces from an external magnetic field, resulting in remotely controllable mechanotransduction. In this investigation, functionalized magnetic nanoparticles were attached to either the mechanically gated TREK1 K+ channel or the (integrin) RGD-binding domains of human mesenchymal stem cells. These cells were microinjected into an ex vivo chick fetal femur (embryonic day 11) that was cultured organotypically in vitro as a model for endochondral bone formation. An oscillating 25-mT magnetic field delivering a force of 4 pN per nanoparticle directly against the mechanoreceptor induced mechanotransduction in the injected mesenchymal stem cells. It was found that cells that received mechanical stimuli via the nanoparticles mineralized the epiphyseal injection site more extensively than unlabeled control cells. The nanoparticle-tagged cells were also seeded into collagen hydrogels to evaluate osteogenesis in tissue-engineered constructs: in this case, inducing mechanotransduction by targeting TREK1 resulted in a 2.4-fold increase in mineralization and significant increases in matrix density. In both models, the combination of mechanical stimulation and sustained release of bone morphogenetic protein 2 (BMP2) from polymer microspheres showed a significant additive effect on mineralization, increasing the effectiveness of BMP2 delivery and demonstrating that nanoparticle-mediated mechanotransduction can be used synergistically with pharmacological approaches for orthopedic tissue engineering to maximize bone formation.